PB1706: ABL-CLASS GENOMIC BREAKPOINT Q-PCR FOR RELIABLE MEASURABLE RESIDUAL DISEASE MONITORING IN ALL PATIENTS
Authors: Inge van Outersterp, Vincent van der Velden, Patricia Hoogeveen, Goda Vaitkevičienė, Gijs van Haaften, Roland Kuiper, Udo Zur Stadt, Judith M. Boer, Monique den Boer
Journal: HemaSphere (2023)

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Approximately 3% of children and 5% of adults who are newly diagnosed with acute lymphoblastic leukemia (ALL) have ABL-class fusions involving tyrosine kinase genes ABL1, ABL2, PDGFRB, and CSF1R. Currently, patients with ABL-class ALL are treated with imatinib, which requires precise monitoring of residual disease. Measurable residual disease (MRD) is typically monitored based on immunoglobulin and T-cell receptor (IG/TR) rearrangements, but this method has limitations, as 5% of patients lack identifiable IG/TR targets, and ongoing rearrangements and oligoclonality can lead to loss of selected IG/TR gene rearrangement. Additionally, this method is not effective in detecting CML-like disease. Aims: We aim to improve response monitoring of ABL-class patients, therefore we developed patient-specific genomic breakpoint Q-PCR assays and assessed whether these can overcome the limitations of IG/TR Q-PCR. Methods: We used leftover diagnostic DNA and performed a variety of next-generation sequencing techniques, such as targeted locus amplification (TLA), genomic capture high-throughput sequencing, and fusion detection by gene enrichment (FUDGE), to determine the genomic breakpoints. The patient-specific Q-PCR assays were designed based on the identified genomic breakpoints. The quantitative range, sensitivity, and MRD values on follow-up time points were determined according to the EuroMRD guidelines. Results: Our study involved 21 ABL-class ALL patients, with PDGFRB (50%) being the most frequently fused kinase, followed by ABL1 (36%), CSF1R (9%), and ABL2 (5%). For all patients, we identified genomic breakpoints that spanned up to 150 kb and developed patient-specific Q-PCR assays with a high sensitivity (≤1E-4) and an adequate quantitative range (≤5E-4). The genomic breakpoint assays enabled us to accurately determine MRD values for three patients who lacked clonal IG/TR targets at diagnosis. Our results showed a strong correlation (Spearman correlation rank test p<0.0001, ρ=0.97, n=66, patients=18) between the MRD values obtained using the IG/TR and genomic breakpoint assays for patients with quantifiable MRD results. However, three patients showed discrepant results, with one case (#6) having quantifiable results only with the genomic breakpoint assay due to an outstanding quantitative range (1E-5), while the IG/TR MRD (QR 1E-4) was positive but not quantifiable. Another case (#19) had higher genomic breakpoint levels compared to IG/TR levels due to a subclone of leukemic cells which could not be captured by IG/TR Q-PCR. Lastly, one case (#12) had high levels of genomic breakpoint MRD during treatment while IG/TR MRD was negative suggesting CML-like disease. Summary/Conclusion: Our study demonstrated that it was feasible to identify the genomic breakpoint and develop a patient-specific Q-PCR assay for MRD monitoring for all ABL-class fused patients. The MRD levels measured using the genomic breakpoint and IG/TR Q-PCR assays were highly correlated, indicating that the genomic breakpoint assay is a reliable alternative when IG/TR targets are unavailable or lost during treatment due to oligoclonality. Figure 1: Correlation between IG/TR MRD level and genomic breakpoint MRD level. Comparison between clinical reported MRD based on IG/TR and patient-specific Q-PCR MRD based on genomic breakpoint (n = 66 samples from 18 patients). All samples with discordant MRD results >1 log10 difference (outside the dotted lines) are colored. Pos, NQ: positive but below the quantitative range of the assay.Keywords: Pediatric, B cell acute lymphoblastic leukemia, Minimal residual disease (MRD)